International Journal of Antimicrobial Agents 51 (2018) 365–369

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International Journal of Antimicrobial Agents

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / i j a n t i m i c a g

Effect of hydroxychloroquine on treatment and recurrence of acute

brucellosis: a single-blind, randomized clinical trial
Mohammad Mahdi Majzoobi a, Seyyed Hamid Hashemi a, Mojgan Mamani a,*,
Fariba Keramat a, Jalal Poorolajal b, Hamid Reza Ghasemi Basir c
a Brucellosis Research Centre, Hamadan University of Medical Sciences, Hamadan, Iran
b Department of Epidemiology, School of Public Health, Hamadan University of Medical Sciences, Hamadan, Iran
c
Department of Pathology, Hamadan University of Medical Sciences, Hamadan, Iran

A R T I C L E I N F O A B S T R A C T

Article history: Brucellosis is associated with a high recurrence rate and requires more than one course of standard treat-
Received 7 March 2017 ment; therefore, more research is required to find more effective treatments that lead to prompt recovery,
Accepted 1 August 2017 and reduce the relapse of disease. This single-blind, randomized study was designed to evaluate the effect
Editor: J.-M. Rolain
of the standard treatment for brucellosis in combination with hydroxychloroquine.
A total of 177 patients with acute brucellosis were randomly assigned to one of two treatment groups:
Keywords:
doxycycline-streptomycin (DS) and doxycycline-streptomycin-hydroxychloroquine (DSH). Clinical symp-
Brucellosis
toms and signs, serological tests, and side effects of therapy were compared between the two groups
Hydroxychloroquine
Relapse during the treatment course and at three and six months after the end of drug therapy. Of the 177 pa-
Treatment tients, with a mean age of 40.5 ± 16.9 years, 66.1% were males. The mean duration of clinical signs prior
to admission was 43.4 ± 41.1 days. Appropriate clinical responses, relapse, treatment failure, and adverse
drug reactions were seen in 98.9%, 1.2%, 0.0%, and 12.6% of patients, respectively, in the DSH group vs.
86.7%, 11.6%, 2.3%, and 19.8% of patients, respectively, in the DS group. There were significant differ-
ences in clinical response and relapse rates between the two groups. The addition of hydroxychloroquine
to a doxycycline-streptomycin regimen appears to increase the efficacy of treatment, accelerate improve-
ment of clinical symptoms, and significantly reduce the rate of relapse of brucellosis.
© 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

1. Introduction
Brucellosis is a zoonotic disease that affects a wide range of
animals, including domestic livestock, and may also lead to human
infection: Brucella melitensis, B. abortus, B. suis, B. canis, and B. ceti
have been isolated from human cases in addition to their specific
animal hosts [1–3]. Brucellosis is considered one of the most
common bacterial zoonotic diseases in the world [1], although its
incidence is difficult to compute accurately because of misdiagno-
sis and under-reporting of infection [4]. However, the disease is
endemic throughout the Mediterranean rim and the Middle East,
with an estimated incidence of more than 100 cases per 100 000
person-years in Iraq, Jordan, and Saudi Arabia [5–7], and a similar
incidence in central Asian countries, such as Kyrgyzstan and Azer-
baijan [8,9]. The prevalence of human disease in endemic areas,
including Africa, Asia, Latin America, and the Middle East, is more
than 10 per 100 000 population [9]. The attention to brucellosis by
* Corresponding author. Division of Infectious Diseases, Sina Hospital, Mirzadeh-
Eshghi Street, Hamadan, 65168, Iran.
E-mail address: mojganmamani@gmail.com (M. Mamani).
the health systems in these areas, which include mostly low-
income countries, does not match the high burden of the disease.
Hence, WHO included brucellosis on the list of neglected zoonotic
diseases [1].
As Iran is in the Middle East region, it is assumed to be an
endemic area for brucellosis. The control of disease is a target for
economic development and health care systems in Iran [8]. The in-
cidence of disease in Iran has increased in recent decades due to
the impossibility of its complete eradication in small ruminants, par-
ticularly sheep and goats. Hamadan province, in the western part
of Iran, has the highest incidence rate in the country of 130 per 100
000 population [9].
Brucellosis symptoms in humans are non-specific and vary from
mild, flu-like symptoms to severe complications in the musculo-
skeletal system, nervous system, and heart [10,11].
Brucellosis is a granulomatous disease that may involve all organs,
and long-term treatment with a combination of several types of
drugs is required to achieve a cure [12]. Conventional treatment
regimens for brucellosis recommended by the WHO include a com-
bination of tetracyclines and streptomycin/gentamicin or tetracyclines
and rifampin [13]. A high rate of relapse and resistance to treat-
ment by administration of different drug regimens, particularly dual

https://doi.org/10.1016/j.ijantimicag.2017.08.009
0924-8579/© 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.
366 M.M. Majzoobi et al. / International Journal of Antimicrobial Agents 51 (2018) 365–369
therapy, in patients with brucellosis have been reported from dif-
ferent areas of the world, even after appropriate antibiotic therapy.
Until now, the best response to treatment and the lowest relapse
rate (about 5–7%) has been achieved with the drug combination of
doxycycline-streptomycin [14].
Treatment failure or recurrent infection may be related to the
intracellular nature of Brucella species [15]. According to previous
studies, the acidic environment of phagolysosomes appears to be
essential for initial proliferation and survival of Brucella. Further-
more, an acidic environment plays a role in the virulence of the
disease [16,17]. On the other hand, changes in the cellular acidic
environment and alkalization of phagolysosomes could enhance the
efficacy of antibiotics on intracellular organisms [17–20]. There-
fore, it appears that the adjustment of the acidic intracellular
environment by using alkalizing agents like hydroxychloroquine [20]
could play a role in the improvement of the treatment response to
brucellosis.
According to the high prevalence of brucellosis in our country,
and frequent treatment failures among brucellosis patients who had
received conventional therapies, we designed this clinical trial to
investigate the efficacy of adding hydroxychloroquine to standard
treatment regimens in Hamadan in the west of Iran.
2. Materials and methods
The study was performed in Sina Hospital, a tertiary-care and
referral center located in Hamadan city, the center of Hamadan prov-
ince in the west of Iran. All patients who attended between 21 March
2015 and 20 March 2016 were considered.
Patients were eligible for the study if they were aged at least 18
years and represented the criteria of acute brucellosis, including a
compatible clinical feature with either a positive Brucella serol-
ogy [Wright ≥1/160, 2-mercaptoethanol (2ME) ≥ 1/80] or positive
blood or bone marrow cultures for Brucella. Patients with glucose-
6-phosphate dehydrogenase deficiency, pregnant women, and those
with significant preexisting diseases, including porphyria, psoriasis,
macular degeneration, or other severe hematological, gastrointes-
tinal, or neurological diseases, were excluded from the study.
Tube agglutination tests based on Wright and 2ME (commer-
cial kits from Pasteur Institute of Iran) were carried out on blood
samples, and antibody titer was determined as per manufactur-
er’s instructions. Serum dilutions from 1/20 to 1/1280 were prepared
with 0.5% phenol saline and 0.5 mL Brucella abortus antigen solu-
tion was added to these dilutions and incubated for 24 h at 37 °C.
Antigen control tubes (without adding the serum) were also incu-
bated. A known positive (1/1280) and negative (saline) control were
also included in each run. Tubes of test series were compared with
antigen control tubes for degree of opacity. The last tube with ag-
glutination was considered as reactive. Wright samples with titers
of 1/160 or higher were reported as positive. The 2ME test was con-
ducted in the same way as the Wright test, but antigenic solution
was mixed with 2ME to break the chemical bonds of IgM. 2ME
removed the remaining IgM; therefore, it can differentiate between
acute and chronic phases of the disease and titers of 1/80 or higher
were considered as positive.
All patients, or their legal relatives, provided a written in-
formed consent before randomization. The study was conducted in
accordance with the principles of the Declaration of Helsinki, and
Hamadan University of Medical Sciences institutional ethics com-
mittee approved the study.
Statistical study assumptions included a 1-sided type I error prob-
ability of 0.05 with a power of 0.8. Assuming a 1-sided significance
level of 5% and a power of 80%, a sample size of 88 patients in each
group would be necessary to demonstrate our expected effect. This
assumption was based on the experiences of a recent study in this
area by the same group that showed a relapse and treatment failure
rate of about 9.2% could be expected to be reduced by about 1%, if
patients were treated with a new drug regimen [21].
Patients were randomly assigned to either an intervention or
control group. Randomization was performed centrally by the study
coordinator. Permuted-block randomization was used with a con-
cealed block size of four.
All patients in the control group received standard medica-
tions for treatment of brucellosis, including doxycycline (100 mg
administered orally twice daily for six weeks) plus streptomycin
(1000 mg/day for patients aged less than 60 years and 750 mg/
day for patients aged at least 60 years, administered by intramuscular
injection for three weeks) (DS group). The intervention group re-
ceived standard treatment plus hydroxychloroquine (DSH group).
The dosage of hydroxychloroquine was based on 6.5 mg/kg for
maximum dosage [22]. The drugs were applied in a single-blind
manner, so that patients did not know about the grouping procedure.
The main follow-up of patients during treatment was clinical.
All patients were assessed clinically at the beginning of treatment
(baseline) and at weekly intervals during the treatment period (six
weeks). They were also followed-up clinically and serologically at
the end of therapy, and three and six months after the cessation
of treatment. Treatment failure was indicated by persistent, or wors-
ening of, clinical symptoms, and lack of reduction, or increase, of
2ME titer. Patients who had an initial clinical response, then recur-
rence of disease symptoms accompanied by increase in 2ME titer,
were considered as relapse cases.
Stata software program (Statacorp TX, USA) was used for all sta-
tistical analyses. Differences between treatment groups were
determined using a t-test for continuous variables and a Fisher’s exact
test or chi-square test for proportional data with a significance level
of P < 0.05.
3. Results
A total of 177 patients, 89 in the DSH group and 88 in the DS
group, were enrolled in this study. Thirty patients in each group were
female. The mean age of participants in the DSH and DS groups were
38.5 ± 17.2 and 42.5 ± 16.4 years, respectively. There was no signif-
icant difference between the two groups for either the mean age
(P = 0.118) or sex distribution (P = 0.957). The mean duration of clin-
ical symptoms in all patients was 43.4 ± 41.1 days. Overall, organ
involvement, such as arthritis, spondylitis, and orchitis, was found
in 41 (48%) patients in the DSH group vs. 44 (52%) in the DS group
(P = 0.653). Table 1 shows the comparison of the clinical manifes-
tations between the two groups of patients.
Of the patients in the DSH group, 88 (98.9%) had a favorable clin-
ical response to treatment at the end of the study, and only one
patient needed a change in treatment regimen. There was no ther-
apeutic failure or prolongation of treatment period in this group.
There was a significant difference in rate of clinical response
between the two groups. Clinical response to treatment during the
Table 1
Clinical presentation of patients with brucellosis in the two treatment groups.
Symptoms/clinical syndromes Treatment group
DSH DS P-value
Generalized pain 62 (69.7%) 72 (81.8%) 0.06
Arthralgia 72 (80.9%) 77 (87.5%) 0.229
Headache 36 (40.4%) 41 (46.6%) 0.410
Fever 43 (48.3%) 53 (60.2%) 0.112
Arthritis 24 (27%) 24 (27.3%) 0.936
Spondylitis 19 (21.3%) 19 (21.6%) 0.969
Orchitis 8 (9%) 8 (9.1%) 0.981
DS: doxycycline + streptomycin; DSH: DS + hydroxychloroquine.
 M.M. Majzoobi et al. / International Journal of Antimicrobial Agents 51 (2018) 365–369
Table 2
Comparison of treatment outcome between the two treatment groups.
Treatment group
DSH DS
Clinical response within two weeks 69.3% 26.3%
Clinical response within six weeks 98.9% 86.7%
Fever disappeared within first week 98.9% 88.7%
Adverse reactions 12.6% 19.8%
Therapeutic failure 0.0% 2.3%
Relapse 1.2%. 11.6%
2ME negative after six months 25.8% 12.5%
2ME: 2-mercaptoethanol; DS: doxycycline + streptomycin; DSH:
hydroxychloroquine.
first, second, and third two-week period of therapy was 61 (69.3%),
23 (26.1%), and 4 (4.5%), respectively, in the DSH group, and 20
(26.3%), 40 (52.6%), and 16 (21.1%), respectively, in the DS group
(P = 0.001). In the DS group, treatment was not favorable for five
patients and some of their complaints remained at the end of the
sixth week, although decline in their 2ME titers reflected a satis-
factory response, so it was not a therapeutic failure. However, the
treatment of these five patients was continued with doxycycline for
12 weeks and there was no clinical symptom at the end of the 12th
week of treatment. In five patients the treatment regimen changed
due to the drug side effect.
There were no patients with therapeutic failure in the DSH group
vs. two patients (2.3%) in the DS group (P = 0.553). Disease relapse
occurred in one patient (1.2%) six months after treatment cessa-
tion in the DSH group compared with in ten patients (11.6%) in the
DS group, including one patient at the first, three patients at the
third, and six patients at the sixth month after the end of treat-
ment (P = 0.004).
A total of 28 patients (15.8%) had an adverse drug reaction, with
11 (12.6%) in the DSH group vs. 17 (19.8%) in the DS group (P = 0.203).
Doxycycline was associated with more photosensitivity reactions
(4 cases), teeth staining (2 cases), nail discoloration (4 cases), der-
matitis (3 cases), and severe epigastric discomfort (4 cases). The
adverse events due to streptomycin were mild hearing loss (1 case),
dizziness and loss of balance (7 cases), and tinnitus (2 cases). Der-
matological complications included itching (2 cases), erythematous
macular and petechial skin rash (2 cases), and erythema multiform
(1 case), which seemed to be due to streptomycin administration.
All the affected patients experienced these reaction symptoms after
the end of treatment with streptomycin (the third week), and healed
at the third month of follow-up, except one patient, whose dizzi-
ness and imbalance symptoms occurred in the second week of
treatment and led to cessation of streptomycin therapy.
An elderly hypertensive patient in the DSH group developed
blurred vision in the left eye after five weeks. Eye examination by
an ophthalmologist revealed optic nerve atrophy in this patient.
Despite discontinuing hydroxychloroquine for this patient, we ob-
served a similar pattern in the right eye after two months. After that,
no worsening of symptoms was observed within six months after
the treatment and the patient did not refer to the provider for
follow-up.
Table 2 shows the characteristics of treatment outcome in the
two treatment groups of the study. Table 3 shows the mean values
of serological tests in both treatment groups at baseline, and the
6th, 12th, and 24th week of evaluation. The mean value of 2ME titer
was significantly lower in the DSH group compared with the DS
group at the 12th and 24th week of assessment.
4. Discussion
The current study was conducted to evaluate the efficacy of
adding hydroxychloroquine to doxycycline-streptomycin combination
367
Table 3
Comparison of 2ME test results between the two treatment groups.
titer decline to ≤ 1/2 of titer decline to ≤ 1/4 of
primary titer primary titer
P-value
WK DHS DS P-value DHS DS P-value
0.001
0.015 6 67(75.28%) 46(52.27%) 0.001 11(12.35%) 11(12.50%) 0.977
0.004 12 80(89.80%) 68(77.27%) 0.023 57(64.04%) 38(43.18%) 0.005
0.203 24 88(98.8%) 66(75%) 0.001 75(84.26%) 57(64.77%) 0.003
0.553
2ME: 2-mercaptoethanol; DS: doxycycline + streptomycin; DSH: DS +
0.004
hydroxychloroquine; WK: Week.
0.024
DS +
therapy in two groups of patients with acute brucellosis in Hamadan
province, west of Iran.
Musculoskeletal features were the major presentation in our
study, and the most local disease involvements were arthritis and
spondylitis. This disease presentation and involvement is sup-
ported by our previous study on 219 brucellosis patients [21].
The rate of response to treatment in the present study indicated
that the patients in the DSH group had a higher and faster response
rate than those in the DS group. Better clinical responses in the DSH
group could be due to changes in the cellular acidic environment
and alkalization of phagolysosome using hydroxychloroquine [18,20].
Some studies have reported an increased antimicrobial activity of
aminoglycosides (streptomycin and amikacin) in the alkaline en-
vironment [17,19]. Although this is not definitely observed in the
case of doxycycline, the synergy between doxycycline and strep-
tomycin is noted in alkaline pH [17], and this could be another reason
for the better clinical outcomes of this treatment regimen. On the
other hand, there could be cross-effects and synergy due to the con-
current administration of doxycycline and hydroxychloroquine [23].
Similarly, in the study by Rault et al., the use of hydroxychloroquine
with doxycycline in the treatment of Q fever endocarditis led to
better treatment outcomes [20].
The anti-inflammatory effect of hydroxychloroquine may reduce
symptoms such as pain and arthritis, but the lower rate of failure
or relapse and the fast decline in 2ME titer in the DSH group strongly
reflected the better response to treatment. Solera et al. [24] con-
ducted a study in 52 patients to assess the efficacy of gentamicin
and doxycycline therapy for human brucellosis. After six months’
follow-up, the relapse rate was 17.3% of patients. In our area, Ranjbar
et al. [25] compared doxycycline-rifampin to triple therapy with
doxycycline-rifampin-amikacin, and they found a similar relapse rate
between the two groups after six months’ follow-up (9.3% in two-
drug regimen vs. 5.7% in triple therapy group). They concluded that
the triple therapy had a higher efficacy and more rapid action in
terms of relief of symptoms compared with the two-drug regimen
[25]. A study by Hashemi et al. from Iran [21] had a therapeutic
failure rate of 8.9% and a relapse rate of 9.0% among all patients
treated with three different drug regimens. Moreover, in a study of
144 patients by Andriopoulos et al. in Greece [26], the rate of treat-
ment failure was 3%, and only four patients who were not compliant
with treatment showed relapse of disease during the follow-up
period. Beside the variations in drug regimen, another possible ex-
planation for these discrepancies in treatment outcome may be
related to the prevalence of different Brucella species; the re-
sponse to treatment in geographical areas where Brucella abortus
is more prevalent may be better compared to those infected with
Brucella melitensis. However, studies from our area indicated the high
rate of treatment failure and relapse with current standard treat-
ment regimens [21,25].
Many adverse effects of brucellosis treatment have been re-
ported in different studies. Solera et al. reported mild adverse events
with a high rate of 36.5% for their treatment protocol (doxycycline
and gentamicin), including photosensitivity, rash, epigastric dis-
comfort, nausea, vomiting, heartburn, anorexia, and erythematous
368 M.M. Majzoobi et al. / International Journal of Antimicrobial Agents 51 (2018) 365–369
rash on the palms, which were mostly related to doxycycline use
[24]. The study by Ranjbar et al. demonstrated mild side effects, such
as gastric complaints, vomiting, phototoxicity, and genital candi-
diasis, in 4.5% of patients treated with doxycycline-rifampin with
and without amikacin regimens, and none of them required dis-
continuation of treatment [25]. Similar adverse events, including
gastrointestinal effects, photosensitivity, dizziness, rash, circumoral
paresthesia, and urticaria, were reported by Hashemi et al [21] in
16.8% of patients treated with doxycycline-streptomycin, doxycycline-
rifampin or ofloxacin-rifampin regimens.
In the present study, development of blurred vision and optic
nerve atrophy in an elderly hypertensive patient led to discontinu-
ation of hydroxychloroquine therapy. Retinopathy is one of the
potential side effects of hydroxychloroquine. Ocular toxicity of
hydroxychloroquine is an uncommon phenomenon and mostly as-
sociated with high daily dosage or long duration of administration
[27]. In some studies, the likelihood of this side effect has been re-
ported to be higher in elderly patients [28]. At a hydroxychloroquine
dose of 400 mg/day, ocular side effects may occur 18 months after
treatment [28]. None of these scenarios applied to our patient. On
the other hand, in cases of brucellosis, eye-related side effects might
occur rarely. Ocular complications of this disease present mainly
as uveitis [29,30], although the involvement of the optic nerve has
been reported [29]. Also eye complications could be due to optic
nerve involvement as a side effect of hypertension [31].
Nail brown-black discoloration induced by doxycycline disap-
peared during the follow-up period, which indicated the reversibility
of this side effect after cessation of therapy [32].
Teeth yellowing following treatment with doxycycline is more
expected in children; however, we observed this side effect in some
adult patients, as has been reported previously [33].
Dizziness and imbalance occurred in elderly patients at the end
of treatment with streptomycin (the third week), apart from in one
patient whose symptoms led to drug change at the second week.
However, dizziness and imbalance disappeared after the third month
of follow-up in all cases. These events occurred in patients whose
daily dose of streptomycin was reduced to 750 mg based on the rec-
ommended dosage in patients aged over 59 years [34]. This may
indicate that streptomycin vestibular ototoxicity is more common
among elderly patients, the effect is slowly reversible, and most of
the effects were at the end of aminoglycoside therapy. Therefore,
the drug should be administered with caution in elderly patients
or even avoided if there is a suitable substitution [35].
In addition to variation in medication regimens, the differ-
ences between treatment-related statistics, particularly reporting
the side effects, may be related to the level of accuracy in tracking
and recording the events.
5. Conclusion
Concurrent administration of doxycycline-streptomycin and
hydroxychloroquine for the treatment of brucellosis may acceler-
ate the improvement of clinical symptoms, reducing failures and
relapses in patients. Although the exact mechanism of this com-
bination therapy in brucellosis is not clear, and this is a limitation
for our study, the improved clinical responses might be due to:
• Acidic environment helps the survival and initial proliferation
and virulence of Brucella, and change of the intracellular envi-
ronment to alkaline could help the treatment by creating a hostile
environment for the disease agent.
• Increase of streptomycin effect in alkaline environments.
• Effect of synergism between doxycycline and streptomycin in al-
kaline cellular environment.
• Concurrent administration of hydroxychloroquine could inten-
sify the effect of doxycycline.
Finally, further studies with large cohorts are warranted to
confirm the results of the present study, and to determine the real
efficacy of this combination therapy for brucellosis.
Acknowledgments
This study was supported by the Vice-chancellor of Research
and Technology, Hamadan University of Medical Sciences, Hamadan,
Iran under the number 9305142390.
Funding: No funding.
Competing interests: None.
Ethical approval: The study protocol was approved by the Ethics
Committee of the Hamadan University of Medical sciences.
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